Not Applicable
Not Applicable
The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode NHL, a putative DNA helicase. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding NHL, substantially purified forms of associated NHL, associated mutant proteins, and methods associated with identifying compounds which modulate NHL, which will be useful in the treatment of various neoplastic disorders, given that this gene is located at 20q13.3 and immediately adjacent to M68/DcR3, which is involved in tumor growth. Also included within the present invention is a human genomic fragment representing this portion of the human genome, along with three additional genes (M68/DcR3, SCLIP, and ARP).
Naumovski et al. (1985, Mol. Cell Biol. 5:17-26; Reynolds et al. (1985 Nucleic Acid Res 13:2357-2372) and Weber et al. (1990 EMBO J. 9:1437-1447) disclose members of the RAD3/FRCC2 gene family of DNA helicases.
It is known that several chemotherapeutic agents inhibit helicases, including actinomycin C1, daunorubicin and nogalamycin (Tuteja, et al., 1997, Biochem. Biophys. Res. Comm. 236(3):636-640), and a prostate cancer drug, C1-958 (Lun, et al., 1998, Cancer Chemother. Pharmacol. 42(6):447-453). In addition, some topoisomerases have been shown to have anti-cancer activity.
Despite the identification of the aforementioned helicase-encoding genes and chemotherapeutic agents, it would be advantageous to identify additional genes which reside within chromosomal regions associated with a disease state such as cancer as well as a gene which encodes a type of protein which may be associated with that disease. The present invention addresses and meets this need by disclosing a DNA molecule encoding a DNA helicase with a chromosomal location suggestive of association with cancer.
The present invention relates to an isolated or purified nucleic acid molecule (polynucleotide) which encodes a novel mammalian DNA helicase.
The present invention also relates to an isolated nucleic acid molecule (polynucleotide) which encodes mRNA which expresses a novel human DNA helicase, NHL.
A preferred aspect of the present invention relates to an isolated or purified DNA molecule which encodes human NHL, the nucleotide sequence as set forth in FIGS. 1A-B and SEQ ID NO: 1.
The present invention also relates to biologically active fragments or mutants of SEQ ID NO: 1 which encode a mRNA molecule expressing a novel DNA helicase, NHL. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the biological properties of the human NHL protein disclosed herein in FIG. 2 and as set forth as SEQ ID NO: 2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a functional NHL protein in a host cell, so as to be useful for screening for agonists and/or antagonists of NHL activity.
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
The present invention also relates to a substantially purified form of a human NHL protein which comprises the amino acid sequence disclosed in FIG. 2 and set forth as SEQ ID NO: 2.
A preferred aspect of this portion of the present invention is a NHL protein which consists of the amino acid sequence disclosed in FIG. 2 and set forth as SEQ ID NO: 2.
Another preferred aspect of the present invention relates to a substantially purified NHL protein, preferably a human NHL protein, obtained from a recombinant host cell containing a DNA expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 1 and expresses the respective NHL protein. It is especially preferred is that the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.
The present invention also relates to biologically active fragments and/or mutants of a NHL protein comprising the amino acid sequence as set forth in SEQ ID NO: 2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators, including but not limited to agonists and/or antagonists for human NHL pharmacology.
A preferred aspect of the present invention is disclosed in FIG. 2 and is set forth as SEQ ID NO: 2, a respective amino acid sequence which encodes human NHL. Characterization of one or more of these DNA helicase-like proteins allows for screening methods to identify novel NHL modulators that may be useful in the treatment of human neoplastic disorders. The modulators selected through such screening and selection protocols may be used alone or in conjunction with other cancer therapies. As noted above, heterologous expression of a NHL protein will allow the pharmacological analysis of compounds which modulate NHL activity and hence may be useful in various cancer therapies. To this end, heterologous cell lines expressing a NHL protein can be used to establish functional or binding assays to identify novel NHL modulators.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the NHL or a biologically active fragment of NHL.
The present invention relates to transgenic mice comprising altered genotypes and phenotypes in relation to NHL and its in vivo activity.
The present invention also relates to NHL fusion constructs, including but not limited to fusion constructs which express a portion of the NHL protein linked to various markers, including but in no way limited to GFP (Green fluorescent protein), the MYC epitope, and GST. Any such fusion constructs may be expressed in the cell line of interest and used to screen for NHL modulators.
Therefore, the present invention relates to methods of expressing mammalian NHL, and preferably human NHL, biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these proteins, and compounds identified through these assays which act as agonists or antagonists of NHL activity.
The present invention also relates to the isolated genomic sequence which comprises SEQ ID NO: 1, a 115 kb genomic fragment set forth herein as SEQ ID NO: 3. As especially preferred aspect of this portion of the invention is the region of the genomic fragment of SEQ ID NO: 3 which comprises the regulatory and coding regions of human NHL, as well as intervening sequences (introns). This 115 kb fragment contains at least the coding region of four genes, NHL, M68/DcR3, SCLIP and ARP. As discussed herein, it has been shown that this region of chromosome 20 is associated with tumor growth. Therefore, an aspect of this invention also comprises the use of one or more regions of this 115 kb genomic sequence to identify compounds which up or downregulate expression of one or more of the genes localized within this 115 kb region, wherein this up or down regulation results in an interference of tumor growth. For example, a transcription element of one of these four genes may be responsible for M68/DcR3 (and/or NHL) overexpression in tumors, and if M68 or NHL overexpression in tumors has a caustic role, blockage of M68/DcR3 or NHL overexpression in tumors by interfering with this transcription site will be useful.
It is an object of the present invention to provide an isolated nucleic acid molecule (e.g., SEQ ID NO: 1) which encodes novel form of human NHL, or fragments, mutants or derivatives of human NHL as set forth in FIG. 2 and SEQ ID NO: 2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators of human NHL activity.
It is a further object of the present invention to provide the mammalian, and especially human, NHL proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding mammalian, and especially human, NHL protein and biological equivalent thereof.
It is an object of the present invention to provide a substantially purified form of human NHL, as set forth in FIG. 2 and SEQ ID NO: 2.
Is another object of the present invention to provide a substantially purified recombinant form of a NHL protein which has been obtained from a recombinant host cell transformed or transfected with a DNA expression vector which comprises and appropriately expresses a complete open reading frame as set forth in SEQ ID NO: 1, resulting in a functional, processed form of NHL. It is especially preferred is that the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.
It is an object of the present invention to provide for biologically active fragments and/or mutants of mammalian, and especially human, NHL, such as set forth in SEQ ID NO: 2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic and/or prophylactic use.
It is also an object of the present invention to use NHL proteins or biological equivalent to screen for modulators, preferably selective modulators, of human NHL activity. Any such compound may be useful in screening for and selecting compounds active against human neoplastic disorders.
As used herein, xe2x80x9csubstantially free from other nucleic acidsxe2x80x9d means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids. Thus, a human NHL DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-NHL nucleic acids. Whether a given NHL DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.
As used herein, xe2x80x9csubstantially free from other proteinsxe2x80x9d or xe2x80x9csubstantially purifiedxe2x80x9d means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins. Thus, a NHL protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-NHL proteins. Whether a given NHL protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting. As used interchangeably with the terms xe2x80x9csubstantially free from other proteinsxe2x80x9d or xe2x80x9csubstantially purifiedxe2x80x9d, the terms xe2x80x9cisolated NHL proteinxe2x80x9d or xe2x80x9cpurified NHL proteinxe2x80x9d also refer to NHL protein that has been isolated from a natural source. Use of the term xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d indicates that NHL protein has been removed from its normal cellular environment. Thus, an isolated NHL protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally. The term isolated does not imply that an isolated NHL protein is the only protein present, but instead means that an isolated NHL protein is substantially free of other proteins and non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the NHL protein in vivo. Thus, a NHL protein that is recombinantly expressed in a prokaryotic or eukaryotic cell and substantially purified from this host cell which does not naturally (i.e., without intervention) express this protein is of course xe2x80x9cisolated NHL proteinxe2x80x9d under any circumstances referred to herein. As noted above, a NHL protein preparation that is an isolated or purified NHL protein will be substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-NHL proteins.
As used interchangeably herein, xe2x80x9cfunctional equivalentxe2x80x9d or xe2x80x9cbiologically active equivalentxe2x80x9d means a protein which does not have exactly the same amino acid sequence as naturally occurring NHL, due to alternative splicing, deletions, mutations, substitutions, or additions, but retains substantially the same biological activity as NHL. Such functional equivalents will have significant amino acid sequence identity with naturally occurring NHL and genes and cDNA encoding such functional equivalents can be detected by reduced stringency hybridization with a DNA sequence encoding naturally occurring NHL. For example, a naturally occurring NHL disclosed herein comprises the amino acid sequence shown as SEQ ID NO: 2 and is encoded by SEQ ID NO: 1. A nucleic acid encoding a functional equivalent has at least about 50% identity at the nucleotide level to SEQ ID NO: 1.
As used herein, xe2x80x9ca conservative amino acid substitutionxe2x80x9d refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
As used herein, the term xe2x80x9cmammalianxe2x80x9d will refer to any mammal, including a human being.